Pol-? catalyzes replication of the genome and other DNA synthetic processes in human cells. However, the precise functions of Pol-? in replication, in repair of endogenous and environmental damage, and in recombination remain to be defined. Further, we lack information on the role of somatic mutations in Pol-? in tumorigenesis and other disease processes. Our goals are to identify the functions of Pol-? in DNA synthesis and the possible role of increased mutation by Pol-? in the generation of human cancer. A major approach will be to exploit mutants of Pol-? that incorporate mutagenic nucleotide analogs and thereby serve as tools to identify DNA synthesized by Pol-? in vivo. We have four specific aims.
In Aim 1, we will generate mutants of Pol-? that increase incorporation of a specific nucleotide analog(s).
In Aim 2, we will purify wild-type and mutant Pol-? holoenzyme complexes and characterize their catalytic properties in detail, including their fidelity and kinetics of analog incorporation.
In Aim 3, we will define the roles of Pol-? in mammalian cells by introducing mutant Pol-?'s that preferentially incorporate mutagenic nucleotide analogs and measuring induced mutation in cells undergoing DNA replication, repair and recombination. The induced mutations will identify the DNA synthesized by Pol-?.
In Aim 4, we will assess the role of increased mutagenesis in tumor progression by performing serial transfer experiments to determine if mammalian cells that harbor mutator Pol-? have a competitive advantage, and if there are nucleoside analogs that diminish this advantage. Narrative Our objective is to establish the roles of DNA polymerase-? in replication of the human genome and in repair of damage caused by endogenous and environmental agents. We will determine if mutations in DNA polymerase-? promote genetic instability and accelerate tumor progression in model systems.
We aim to identify a new class of chemotherapeutic agents that will retard tumor growth.
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|Prindle, Marc J; Schmitt, Michael W; Parmeggiani, Fabio et al. (2013) A substitution in the fingers domain of DNA polymerase Î´ reduces fidelity by altering nucleotide discrimination in the catalytic site. J Biol Chem 288:5572-80|
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|Schmitt, Michael W; Kennedy, Scott R; Salk, Jesse J et al. (2012) Detection of ultra-rare mutations by next-generation sequencing. Proc Natl Acad Sci U S A 109:14508-13|
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